Imaging Intracellular H₂O₂ with the Genetically Encoded PerFRET and OxyFRET Probes

Hydrogen peroxide (H₂O₂) is an important signaling intermediate with various regulatory and effector functions. Despite its significance, the subcellular organization of H₂O₂ signals is poorly understood. Introducing novel techniques for the intracellular detection of H₂O₂ would be essential for a more complete understanding of its role in cellular signaling. We previously reported the development of two novel fluorescence resonance energy transfer (FRET)-based protein sensors that showed opposite emission ratio changes upon reaction with H₂O₂. In this chapter, we detail the methods for using OxyFRET and PerFRET for the assessment of changes in subcellular H₂O₂ levels.